A Novel Arsenate-Resistant Determinant Associated with ICEpMERPH, a Member of the SXT/R391 Group of Mobile Genetic Elements

ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its genetic structure, and its homology to related elements normally found in pathogenic Vibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The “hotspot regions” of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes: orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of Escherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1–5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

SXT/R391 ICEs with ** found in the study by (Bioteau et al. 2018), ICEs are scattered over two contigs extracted WGS data, an estimation of the minimum size is provided.

*Unpublished Sequence data ** Not annotated or partially annotated
Abbreviations associated with this table: AAA: ATPases Associated with diverse cellular activities , Ac r : Acriflavin resistance , Af r : Actiflavine resistance, Ag r : Aminoglycoside resistance, Am r : Ampicillin resistance, Amk r :Amikacin resistance, Ap r : Apramycin resistance, As r : Arsenic resistance, Azm r : Azithromycin resistance, Az r : Aztreonam resistance, A/S r : Ampicillin-sulbactam resistance, Bm r -Bicyclomycin resistance, Supplementary Figure S1. Common arsenic resistant systems located on the E. coli chromosome, on E. coli plasmids R773 and R46 and on Staphylococcus aureus plasmids pI258 and pSX267, a novel arsenic resistant system found on the transposon TnAtcArs, the ICE mobile genetic ICESde3369, the resistant system found on the chromosome in P. aeruginosa DK2 and the arsenic resistant system found in ICEpMERPH.
Comparative analysis was carried out to compare the arsenic resistant system found in HS4 of ICEpMERPH with other well studied arsenic resistant systems. This should give an insight into how this system differs from other well-studied arsenic resistant systems. Well studied arsenic systems are found on the chromosome of certain Escherichia coli and in bacterial mobile genetic elements. All these systems have similar molecular gene structure, the E. coli encoded arsenic operon contains genes arsRBC [51] and the same arrangement is observed in both plasmids contained in gram-positive Staphylococcus aureus pI258 (RBC) [52] and Staphylococcus xylosus pSX267 (RBC) [53].
Similarly, the plasmids R773 and R46 share three of the same genes with the addition of arsA and arsD [54,55]. arsA and arsD are missing from the E. coli operon and the Staphylococcus arsenic operons. ArsD is a secondary regulator and has apparently no effect on the level of resistance. arsA is more important, it's a membrane transporter that can switch energy coupling modes from chemiosmotic or an ATPase pump [56].
All these well studied arsenic operons are mapped in Figure S1. The number of genes can vary between the gram-negative and gram-positive organisms and the details of their functions can also vary. R773 and R46 protein products share 85 % and 93% similarity [56] while those associated with pI258 and pSX267 share 93 % amino acid identity [56]. Plasmid associated arsenic resistant systems have been well studied but there is little information on systems in other MGE or ICEs which may have changed or evolved.
An unusual novel arsenic resistant transposon, TnAtcArs, was identified in the bacterium Acidithiobacillus caldus [57], its gene arrangement is different from any other system identified containing Ars RCDADA [57]. Another novel arsenic resistance system was also identified in an ICE, ICESde3369 (EU142041) which was unlike other arsenic resistance systems so far discovered [58] ( Figure S1).
The predicted ICEpMERPH arsenic resistance system contained in HS4 contains 7 genes, the last three genes encoding homologs of ArsP, ACR3 and ArsR. The ArsP-like proteins are permeases similar to a methylarsenite efflux permease found in Camylobacter jejuni [59]. This was the first identified efflux system specific for trivalent organarsenicals [59]. The putative protein ACR3 is a homolog of an arsenite transporter that demonstrated resistance to arsenate but not arsenite in arsenic hypersensitive E. coli AW3110 [60]. The putative ArsR protein is a homolog of an arsenic inducible repressor [61] that is commonly seen in arsenic resistance determinants. This system is missing an arsenic reductase, and thus it does not seem to be a complete operon and therefore may not be fully functional compared to full operon containing systems and may have changed by insertion or deletion of genes over time. Putative tyrosine phosphate and thioredoxin protein encoding genes appear before these putative Ars genes but there is as yet no evidence that they have any relation to arsenic resistance.
It may be an adaptive advantage that this putative system is missing ArsC, the arsenate reductase enzyme. Arsenate reductases reduces the less toxic arsenate (As(III)) to the more toxic arsenite (As(III)) [52] where conversion of a less toxic compound to a more toxic compound seems counter-productive [56]. This may explain the two-gene system at the beginning of the hotspot, similarly found on the chromosome of Pseudomonas aeruginosa DK2 which confers resistance to arsenate. Detoxification systems like this may have evolved early when arsenic was reduced to As(III). This system does not appear to require cellular reductants or reductases and it does not require biotransformation to more reactive and toxic trivalent species [62] which is a major adaptive advantage.
The ICEpMERPH system and that found in P. aeruginosa DK2 are illustrated in Figure S1. Which illustrates that the two systems are different in gene synteny and orientation. P. aeruginosa DK2 contains known arsenic resistant genes such as arsR, arsC, acr3 and arsH which are different to the system found in ICEpMERPH, the only similarity being the two-gene system that confers resistance to arsenate.
Thus, we hypothesise that the ICEpMERPH system is a novel arsenate efflux system not seen in other SXT/R391 ICE's or in any other bacterial mobile element to the best of our knowledge.